MHC class I proteins (MHC class I molecules, Major Histocompatibility Complex Class I molecules, Major Histocompatibility Complex Class I proteins) are transmembrane proteins of the cellular immune response, which bind antigenic peptides from the cell interior of an antigen-presenting cell (APC), such as e.g., from the cytosol or the lumen of endocytic organelles, and present them to the cytotoxic T-cells (CTL, cytotoxic T lymphocytes, killer T cells, CD8 positive lymphocytes) at the cell surface. This is referred to as a so-called class I antigen presentation.
Binding a T-cell receptor (TCR) of a CTL to a class I peptide complex of an APC leads to the activation of the CTL and/or to the induction of cell death (apoptosis) of the APC by the CTL (depending on the location of the reaction in the body, the type of APC (B-cell, dendritic cell, etc.) and the activation state of the CTL).
The specificity (ability to react with a certain class I peptide complex) of a CTL is based on the fact that it carries only one type of TCR (i.e., only TCRs of a single distinct sequence). The immune response is effective because such CTLs, which react with “self”-peptides (peptides produced from the body's own proteins) are eliminated in the thymus.
For this reason, the identification of an antigenic peptide by the TCR of a CTL means that the APC produces foreign proteins, which could stem from viruses or intracellular parasites (bacteria, protozoa). An overproduction of endogenous peptides in malignant degenerated cells, e.g., in tumors, can also lead to such identification reactions.
Almost all proteins present in the cell are broken down into peptides, which subsequently bind with MHC class I proteins in the endoplasmic reticulum (an organelle enclosed by a membrane inside the cell). The complex formed by the peptide and the MHC class I protein (herein referred to as class I peptide complex) is subsequently transported to the surface of the cell where it is available for identification by CTLs.
If novel or mutated proteins are produced due to a tumorigenic malignant degeneration, or if viral or bacterial proteins are produced from the genetic material of a virus or bacteria in case of a viral or bacterial infection, these “novel” proteins are also broken down into peptides, which are then presented in a complex with MHC class I proteins at the cell surface. These “novel” peptides are different from the cell's own peptides and trigger identification by the CTL.
The presentation by MHC class I proteins is also important in allergic reactions, the rejection of transplants, and a number of autoimmune diseases such as multiple sclerosis and spondyloarthropathies (Bechterew's disease or rheumatoid arthritis).
The examination of the immune responses mediated by MHC class I proteins often requires detecting the CTLs that react with a specific class I peptide complex (epitope). Reactions to a single immunodominant epitope often represent 10-20% of the total T-cell population in an organism, so that tracing the T-cell frequency (i.e., the share of CTLs that react with a specific epitope in the total amount of CTLs in the organism) allows following precisely the immune response (e.g., the success or failure of a therapy). Reagents that can identify the epitope specificity of CTLs are therefore indispensable.
Recombinant MHC class I proteins are currently used to detect such epitope-specific CTLs. These proteins are produced in bacteria and are provided as insoluble inclusion bodies that are first denatured in a solution of a chaotropic agent. The chaotropic agent is then removed in the presence of the desired peptide and the class I peptide complex is separated from the unfolded protein by gel filtration chromatography (if necessary). Since the low affinity of a single class I peptide complex with a single TCR does not lead to a tight binding, multimers of class I peptide complexes are used, which, due to the avidity effect, bind tightly enough with the TCR of a CTL to allow for a durable bond.
Such multimers are obtained, for example, by streptavidin-mediated tetramerization of biotinylated class I peptide complexes (class I tetramers), by pentamerization by self-assembling coiled-coil domains (class I pentamers), or by multimerization on dextran (class I dextramers). There are other methods for oligomerization or multimerization of class I peptide complexes.
In general, class I multimers are labelled with fluorescent dyes, which allow them to be detected by microscopic or flow cytometric detection. Epitope-specific CTLs can thus be directly colored for the purpose of detection.
Other uses for recombinant class I peptide complexes are:                In vitro—Selection and expansion of monospecific T-cells for reinfusion in case of cancer and viral diseases. The selection can be carried out by cytofluorometry (flow cytometry, FACS), by magnet-activated cell sorting (MACS) or, for an increased throughput, in microarrays.        Ex vivo—Isolation and expansion of CTLs for adoptive therapy after allogeneic stem cell transplant.        Ex vivo—Removal of alloreactive T-cells after peripheral stem cell transplantation. The removal of autoreactive T-cells, which cause type 1 diabetes, arthritis and other autoimmune diseases, is also interesting, as has already been described with regard to MHC class II reagents. Using isotopically labelled multimers is described in US 2003/0228258 A1.        
The production of recombinant class I peptide complexes is complicated, time-consuming and expensive. On the one hand, several thousand MHC class I allotype are known (of which admittedly five alleles of HLA-A cover approximately 50% of the world's population). First and foremost, however, a new multimer must be produced for each antigenic peptide that is to be examined as an epitope so that new multimers, which must be specifically produced as required, are needed for each patient or for each experiment.
It would be simpler to produce the multimers without the antigenic peptides and to subsequently add the respectively required antigenic peptides as necessary. This has not, however, to date been possible because folding MHC class I proteins without an antigenic peptide is not possible for most MHC class I allotypes.
A method involving a special peptide was previously described, where the special peptide is decomposed by a UV treatment or a periodate treatment, and can then be replaced by an added examination peptide (publication: Rodenko et al., Nature Protocols 1 (2006), p. 1120).
This method is expensive, complicated and does not work with all antigenic peptides or class I allotypes.